Clearance of defective muscle stem cells by senolytics reduces the expression of senescence-associated secretory phenotype and restores myogenesis in myotonic dystrophy type 1

Muscle weakness and atrophy are clinical hallmarks of myotonic dystrophy type 1 (DM1). Muscle stem cells, which contribute to skeletal muscle growth and repair, are also affected in this disease. However, the molecular mechanisms leading to this defective activity and the impact on the disease severity are still elusive. Here, we explored through an unbiased approach the molecular signature leading to myogenic cell defects in DM1. Single cell RNAseq data revealed the presence of a specific subset of DM1 myogenic cells expressing a senescence signature, characterized by the high expression of genes related to senescence-associated secretory phenotype (SASP). This profile was confirmed using different senescence markers in vitro and in situ. Accumulation of intranuclear RNA foci in senescent cells, suggest that RNA-mediated toxicity contribute to senescence induction. High expression of IL-6, a prominent SASP cytokine, in the serum of DM1 patients was identified as a biomarker correlating with muscle weakness and functional capacity limitations. Drug screening revealed that the BCL-XL inhibitor (A1155463), a senolytic drug, can specifically target senescent DM1 myoblasts to induce their apoptosis and reduce their SASP. Removal of senescent cells re-established the myogenic function of the non-senescent DM1 myoblasts, which displayed improved proliferation and differentiation capacity in vitro; and enhanced engraftment following transplantation in vivo. Altogether this study presents a well-defined senescent molecular signature in DM1 untangling part of the pathological mechanisms observed in the disease; additionally, we demonstrate the therapeutic potential of targeting these defective cells with senolytics to restore myogenesis

Cardiomyocyte senescence characterization and identification of associated markers

Le vieillissement de l'organisme prédispose à de nombreuses pathologies chroniques telles que l'insuffisance cardiaque (IC). Des études récentes ont montré que l'accumulation de cellules sénescentes dans les organes au cours du vieillissement est associée à l'apparition de ces pathologies. La sénescence cellulaire a initialement été décrite comme un arrêt stable du cycle cellulaire permettant de limiter la prolifération des cellules dont l'ADN est endommagé. Ce processus s'accompagne de profondes modifications de la fonction cellulaire, avec notamment l'acquisition d'un phénotype sécrétoire associé à la sénescence. La sénescence peut être induite par un raccourcissement des télomères ou par l'exposition à des signaux de stress, tels que le stress oxydant ou l'irradiation, qui entrainent l'activation de la réponse cellulaire aux dommages de l'ADN et l'expression des gènes suppresseurs de tumeurs (p16INK4a, p21CIP1, p53). Ces inhibiteurs du cycle cellulaire sont classiquement utilisés comme marqueur de sénescence car leur expression augmente de manière ubiquitaire au cours du vieillissement. Toutefois, ces marqueurs ne sont pas spécifiques du tissu concerné et un des objectifs de ma thèse a été d'identifier de nouveaux marqueurs de sénescence tissu-spécifiques qui pourraient caractériser un vieillissement cardiaque pathologique. Le vieillissement cardiaque se caractérise par une hypertrophie des cardiomyocytes, une sensibilité accrue au stress et une prédisposition à l'IC. Les cardiomyocytes étant des cellules post-mitotiques, les mécanismes de sénescence mis en jeu, les marqueurs associés et leur rôle potentiel dans l'IC demeurent à l'heure actuelle peu caractérisés. Au cours de ce travail de thèse nous avons donc entrepris : 1) d'étudier le rôle des télomères et des dysfonctions mitochondriales dans l'induction de la sénescence du cardiomyocyte et 2) d'identifier des marqueurs spécifiques. Nous avons tout d'abord montré que les cardiomyocytes de souris âgées expriment les marqueurs classiques de la sénescence comme p16INK4a, p53 et p21CIP1. Concernant les mécanismes inducteurs, nous avons étudié l'implication des dommages télomériques (telomere associated foci, TAF). Au cours du vieillissement, nous avons observé une augmentation du nombre de TAFs par cardiomyocytes en association avec l'hypertrophie. De plus, l'induction de TAFs in vitro est suffisante à l'activation de la voie de sénescence p53/p21CIP1 et l'hypertrophie dans une lignée de cardiomyoblastes H9c2. La formation des TAFs est augmentée chez des souris avec une dysfonction mitochondriale et est associée à l'activation des voies p53/p21CIP1. Par ailleurs, les cardiomyocytes âgés présentent une dérégulation des gènes impliqués dans la biologie mitochondriale pouvant rendre compte de l'augmentation des TAFs. Par l'analyse haut débit du transcriptome (RNAseq) nous avons identifié six nouveaux gènes qui sont surexprimés dans les cardiomyocytes sénescents (Prom2, Kcnk1, Pah, Edn3, Gdf15, Tgfb2). La comparaison d'expression de ces gènes dans le cœur avec d'autres tissus et avec le stroma cardiaque lors vieillissement a permis de confirmer la spécificité d'expression de ces marqueurs au niveau des cardiomyocytes. Nous avons validé cette signature dans deux modèles in vitro de sénescence induite par le stress et démontré que l'expression de certains de ces marqueurs est dépendante de la voie p53. De plus, l'expression de Prom2 est associée à l'hypertrophie des cardiomyocytes. En conclusion, nous avons démontré, qu'avec le vieillissement, les cardiomyocytes présentent un programme de sénescence associé à une dysfonction mitochondriale et une augmentation des TAFs. Cette sénescence se caractérise par l'activation des voies classiques de sénescence (p16INK4, p53/p21CIP1), une hypertrophie et l'acquisition d'une signature spécifique. Ces marqueurs offrent de nouvelles perspectives dans la compréhension de la sénescence cardiaque et dans son implication potentielle dans l'IC.Ageing of the organism is associated with several chronic pathologies such as heart failure (HF). Recent studies have demonstrated the link between the accumulation of senescent cells during ageing and age-associated diseases. Cellular senescence, originally defined as a stable cell cycle arrest, acts as a tumorigenic repressor by limiting the proliferation of DNA damaged cells. Despite this protective effect, senescence is characterized by deep remodeling of cell biology which drives functional disorders, such as the acquisition of a senescence-associated secretory phenotype (SASP). Senescence can be induced by telomeric attrition and by exposition to cellular stress signals such as oxidative stress or irradiation, which induce telomeric damage, activation of the DNA Damage Response (DDR) and increased expression of antitumoral genes (p16INK4a, p21CIP1, p53). These genes are classically used as markers of senescence because their expression increases in several tissues during ageing but they are not tissue-specific. Therefore, At the cardiac level, ageing is characterized by cardiomyocytes hypertrophy, increased sensitivity to stress and highest risk of developing HF. Cardiomyocytes are post- mitotic cells and the senescence inductor mechanisms, specifics markers and their role in HF remains poorly understood. This thesis project is articulated around two aims, 1/ studying the role of telomeric damages and mitochondrial dysfunction in triggering cardiomyocyte senescence and 2/ identification of specifics markers. Fisrtly, we shown that aged cardiomyocytes overexpress classic markers of senescence such as p16INK4a, p53 et p21CIP1. Concerning the inductors mechanisms, we studied the implication of telomeric damages (telomere associated foci, TAF). During ageing, we found an increased number of TAFs per cardiomyocytes and their association with hypertrophy. Moreover, TAF- induction in cardiac H9c2 in vitro activated the p53/p21 pathway and induced senescence. These data confirmed the role of TAFs in cardiomyocyte senescence induction. Furthermore, aged cardiomyocytes exhibit a global alteration of genes involved in mitochondrial biology, oxidative stress and metabolism in aged cardiomyocytes that could play a prominent role in TAF accumulation with ageing. In a second part of the study, by using a next generation sequencing method (RNA-seq) we identified 6 new genes highly expressed in senescent cardiomyocytes (Prom2, Kcnk1, Pah, Edn3, Gdf15 and Tgfb2). Expression comparison with other senescent organs and cardiac stromal cells confirmed these new genes as cardiomyocyte specific. Thanks to an in vitro approach, we validate this signature by using different models of stress-induced senescence in cardiac H9c2 cells and demonstrated the implication of the p53 in the regulation of some of these genes. Moreover, Prom2 expression is associated with cardiomyocytes hypertrophy. In conclusion, we demonstrated that, with ageing, cardiomyocytes display a senescence phenotype associated with mitochondrial dysfunction and TAFs. This process is characterized by classic markers (p16INK4, p53/p21CIP1), hypertrophy and new identified signature. These new markers offer innovative perspectives in the understanding and the identification of the cardiac senescence and their potential deleterious role in heart failure

Caractérisation de la sénescence des cardiomyocytes et identification de marqueurs associés

Ageing of the organism is associated with several chronic pathologies such as heart failure (HF). Recent studies have demonstrated the link between the accumulation of senescent cells during ageing and age-associated diseases. Cellular senescence, originally defined as a stable cell cycle arrest, acts as a tumorigenic repressor by limiting the proliferation of DNA damaged cells. Despite this protective effect, senescence is characterized by deep remodeling of cell biology which drives functional disorders, such as the acquisition of a senescence-associated secretory phenotype (SASP). Senescence can be induced by telomeric attrition and by exposition to cellular stress signals such as oxidative stress or irradiation, which induce telomeric damage, activation of the DNA Damage Response (DDR) and increased expression of antitumoral genes (p16INK4a, p21CIP1, p53). These genes are classically used as markers of senescence because their expression increases in several tissues during ageing but they are not tissue-specific. Therefore, At the cardiac level, ageing is characterized by cardiomyocytes hypertrophy, increased sensitivity to stress and highest risk of developing HF. Cardiomyocytes are post- mitotic cells and the senescence inductor mechanisms, specifics markers and their role in HF remains poorly understood. This thesis project is articulated around two aims, 1/ studying the role of telomeric damages and mitochondrial dysfunction in triggering cardiomyocyte senescence and 2/ identification of specifics markers. Fisrtly, we shown that aged cardiomyocytes overexpress classic markers of senescence such as p16INK4a, p53 et p21CIP1. Concerning the inductors mechanisms, we studied the implication of telomeric damages (telomere associated foci, TAF). During ageing, we found an increased number of TAFs per cardiomyocytes and their association with hypertrophy. Moreover, TAF- induction in cardiac H9c2 in vitro activated the p53/p21 pathway and induced senescence. These data confirmed the role of TAFs in cardiomyocyte senescence induction. Furthermore, aged cardiomyocytes exhibit a global alteration of genes involved in mitochondrial biology, oxidative stress and metabolism in aged cardiomyocytes that could play a prominent role in TAF accumulation with ageing. In a second part of the study, by using a next generation sequencing method (RNA-seq) we identified 6 new genes highly expressed in senescent cardiomyocytes (Prom2, Kcnk1, Pah, Edn3, Gdf15 and Tgfb2). Expression comparison with other senescent organs and cardiac stromal cells confirmed these new genes as cardiomyocyte specific. Thanks to an in vitro approach, we validate this signature by using different models of stress-induced senescence in cardiac H9c2 cells and demonstrated the implication of the p53 in the regulation of some of these genes. Moreover, Prom2 expression is associated with cardiomyocytes hypertrophy. In conclusion, we demonstrated that, with ageing, cardiomyocytes display a senescence phenotype associated with mitochondrial dysfunction and TAFs. This process is characterized by classic markers (p16INK4, p53/p21CIP1), hypertrophy and new identified signature. These new markers offer innovative perspectives in the understanding and the identification of the cardiac senescence and their potential deleterious role in heart failure.Le vieillissement de l'organisme prédispose à de nombreuses pathologies chroniques telles que l'insuffisance cardiaque (IC). Des études récentes ont montré que l'accumulation de cellules sénescentes dans les organes au cours du vieillissement est associée à l'apparition de ces pathologies. La sénescence cellulaire a initialement été décrite comme un arrêt stable du cycle cellulaire permettant de limiter la prolifération des cellules dont l'ADN est endommagé. Ce processus s'accompagne de profondes modifications de la fonction cellulaire, avec notamment l'acquisition d'un phénotype sécrétoire associé à la sénescence. La sénescence peut être induite par un raccourcissement des télomères ou par l'exposition à des signaux de stress, tels que le stress oxydant ou l'irradiation, qui entrainent l'activation de la réponse cellulaire aux dommages de l'ADN et l'expression des gènes suppresseurs de tumeurs (p16INK4a, p21CIP1, p53). Ces inhibiteurs du cycle cellulaire sont classiquement utilisés comme marqueur de sénescence car leur expression augmente de manière ubiquitaire au cours du vieillissement. Toutefois, ces marqueurs ne sont pas spécifiques du tissu concerné et un des objectifs de ma thèse a été d'identifier de nouveaux marqueurs de sénescence tissu-spécifiques qui pourraient caractériser un vieillissement cardiaque pathologique. Le vieillissement cardiaque se caractérise par une hypertrophie des cardiomyocytes, une sensibilité accrue au stress et une prédisposition à l'IC. Les cardiomyocytes étant des cellules post-mitotiques, les mécanismes de sénescence mis en jeu, les marqueurs associés et leur rôle potentiel dans l'IC demeurent à l'heure actuelle peu caractérisés. Au cours de ce travail de thèse nous avons donc entrepris : 1) d'étudier le rôle des télomères et des dysfonctions mitochondriales dans l'induction de la sénescence du cardiomyocyte et 2) d'identifier des marqueurs spécifiques. Nous avons tout d'abord montré que les cardiomyocytes de souris âgées expriment les marqueurs classiques de la sénescence comme p16INK4a, p53 et p21CIP1. Concernant les mécanismes inducteurs, nous avons étudié l'implication des dommages télomériques (telomere associated foci, TAF). Au cours du vieillissement, nous avons observé une augmentation du nombre de TAFs par cardiomyocytes en association avec l'hypertrophie. De plus, l'induction de TAFs in vitro est suffisante à l'activation de la voie de sénescence p53/p21CIP1 et l'hypertrophie dans une lignée de cardiomyoblastes H9c2. La formation des TAFs est augmentée chez des souris avec une dysfonction mitochondriale et est associée à l'activation des voies p53/p21CIP1. Par ailleurs, les cardiomyocytes âgés présentent une dérégulation des gènes impliqués dans la biologie mitochondriale pouvant rendre compte de l'augmentation des TAFs. Par l'analyse haut débit du transcriptome (RNAseq) nous avons identifié six nouveaux gènes qui sont surexprimés dans les cardiomyocytes sénescents (Prom2, Kcnk1, Pah, Edn3, Gdf15, Tgfb2). La comparaison d'expression de ces gènes dans le cœur avec d'autres tissus et avec le stroma cardiaque lors vieillissement a permis de confirmer la spécificité d'expression de ces marqueurs au niveau des cardiomyocytes. Nous avons validé cette signature dans deux modèles in vitro de sénescence induite par le stress et démontré que l'expression de certains de ces marqueurs est dépendante de la voie p53. De plus, l'expression de Prom2 est associée à l'hypertrophie des cardiomyocytes. En conclusion, nous avons démontré, qu'avec le vieillissement, les cardiomyocytes présentent un programme de sénescence associé à une dysfonction mitochondriale et une augmentation des TAFs. Cette sénescence se caractérise par l'activation des voies classiques de sénescence (p16INK4, p53/p21CIP1), une hypertrophie et l'acquisition d'une signature spécifique. Ces marqueurs offrent de nouvelles perspectives dans la compréhension de la sénescence cardiaque et dans son implication potentielle dans l'IC

Senescence and Aging: Does It Impact Cancer Immunotherapies?

Cancer incidence increases drastically with age. Of the many possible reasons for this, there is the accumulation of senescent cells in tissues and the loss of function and proliferation potential of immune cells, often referred to as immuno-senescence. Immune checkpoint inhibitors (ICI), by invigorating immune cells, have the potential to be a game-changers in the treatment of cancer. Yet, the variability in the efficacy of ICI across patients and cancer types suggests that several factors influence the success of such inhibitors. There is currently a lack of clinical studies measuring the impact of aging and senescence on ICI-based therapies. Here, we review how cellular senescence and aging, either by directly altering the immune system fitness or indirectly through the modification of the tumor environment, may influence the cancer-immune response

Monoamine Oxidases, Oxidative Stress, and Altered Mitochondrial Dynamics in Cardiac Ageing

International audienceThe advances in healthcare over the past several decades have resulted in populations now living longer. With this increase in longevity, a wider prevalence of cardiovascular diseases is more common and known to be a major factor in rising healthcare costs. A wealth of scientific evidence has implicated cell senescence as an important component in the etiology of these age-dependent pathologies. A number of studies indicate that an excess of reactive oxygen species (ROS) contributes to trigger and accelerate the cardiac senescence processes, and a new role of monoamine oxidases, MAO-A and MAO-B, is emerging in this context. These mitochondrial enzymes regulate the level of catecholamines and serotonin by catalyzing their oxidative deamination in the heart. MAOs' expression substantially increases with ageing (6-fold MAO-A in the heart and 4-fold MAO-B in neuronal tissue), and their involvement in cardiac diseases is supposedly related to the formation of ROS, via the hydrogen peroxide produced during the substrate degradation. Here, we will review the most recent advances in this field and describe why MAOs could be effective targets in order to prevent age-associated cardiovascular disease

Identification of Prominin‐2 as a new player of cardiomyocyte senescence in the aging heart

International audienceThe aging heart is characterized by a number of structural changes leading to ventricular stiffness, impaired resistance to stress and increased risk of developing heart failure (HF). Genetic or pharmacological removal of senescent cells has recently demonstrated the possibility to relieve some cardiac aging features such as hypertrophy and fibrosis. However, the contribution of the different cell types in cardiac aging remains fragmentary due to a lack of cell‐specific markers. Cardiomyocytes undergo post‐mitotic senescence in response to telomere damage, characterized by persistent DNA damage response and expression of the classical senescence markers p21 and p16, which are shared by many other cell types. In the present study, we used transcriptomic approaches to discover new markers specific for cardiomyocyte senescence. We identified Prominin2 (Prom2), encoding a transmembrane glycoprotein, as the most upregulated gene in cardiomyocytes of aged mice compared to young mice. We showed that Prom2 was upregulated by a p53‐dependent pathway in stress‐induced premature senescence. Prom2 expression correlated with cardiomyocyte hypertrophy in the hearts of aged mice and was increased in atrial samples of patients with HF with preserved ejection fraction. Consistently, Prom2 overexpression was sufficient to drive senescence, hypertrophy and resistance to cytotoxic stress while Prom2 shRNA silencing inhibited these features in doxorubicin‐treated cardiac cells. In conclusion, we identified Prom2 as a new player of cardiac aging, linking cardiomyocyte hypertrophy to senescence. These results could provide a better understanding and targeting of cell‐type specific senescence in age‐associated cardiac diseases

Kidney inflammaging is promoted by CCR2+ macrophages and tissue-derived micro-environmental factors

International audienceAbstract The incidence of disorders associated with low inflammatory state, such as chronic kidney disease, increases in the elderly. The accumulation of senescent cells during aging and the senescence-associated secretory phenotype, which leads to inflammaging, is known to be deleterious and account for progressive organ dysfunction. To date, the cellular actors implicated in chronic inflammation in the kidney during aging are still not well characterized. Using the DECyt method, based on hierarchical clustering of flow cytometry data, we showed that aging was associated with significant changes in stromal cell diversity in the kidney. In particular, we identified two cell populations up-regulated with aging, the mesenchymal stromal cell subset (kMSC) expressing CD73 and the monocyte-derived Ly6C + CCR2 + macrophage subset expressing pro-inflammatory cytokines. Aged CD73 + kMSCs depicted senescence associated features with low proliferation rate, increased DNA damage foci and Ccl2 expression. Using co-cultures experiments, we showed that aged CD73 + kMSC promoted monocyte activation and secretion of inflammatory cytokines albeit less efficiently than young CD73 + kMSCs. In the context of ageing, increased frequency of CD73 + kMSC subpopulations could provide additional niche factors to newly recruited monocytes favoring a positive regulatory loop in response to local inflammation. Interfering with such partnership during aging could be a valuable approach to regulate kidney inflammaging and to limit the risk of developing chronic kidney disease in the elderly

Restored immune cell functions upon clearance of senescence in the irradiated splenic environment

Some studies show eliminating senescent cells rejuvenate aged mice and attenuate deleterious effects of chemotherapy. Nevertheless, it remains unclear whether senescence affects immune cell function. We provide evidence that exposure of mice to ionizing radiation (IR) promotes the senescent-associated secretory phenotype (SASP) and expression of p16(INK4a) in splenic cell populations. We observe splenic T cells exhibit a reduced proliferative response when cultured with allogenic cells in vitro and following viral infection in vivo. Using p16-3MR mice that allow elimination of p16(INK4a)-positive cells with exposure to ganciclovir, we show that impaired T-cell proliferation is partially reversed, mechanistically dependent on p16(INK4a) expression and the SASP. Moreover, we found macrophages isolated from irradiated spleens to have a reduced phagocytosis activity in vitro, a defect also restored by the elimination of p16(INK4a) expression. Our results provide molecular insight on how senescence-inducing IR promotes loss of immune cell fitness, which suggest senolytic drugs may improve immune cell function in aged and patients undergoing cancer treatment

Selective Cardiomyocyte Oxidative Stress Leads to Bystander Senescence of Cardiac Stromal Cells

International audienceAccumulation of senescent cells in tissues during normal or accelerated aging has been shown to be detrimental and to favor the outcomes of age-related diseases such as heart failure (HF). We have previously shown that oxidative stress dependent on monoamine oxidase A (MAOA) activity in cardiomyocytes promotes mitochondrial damage, the formation of telomere-associated foci, senescence markers, and triggers systolic cardiac dysfunction in a model of transgenic mice overexpressing MAOA in cardiomyocytes (Tg MAOA). However, the impact of cardiomyocyte oxidative stress on the cardiac microenvironment in vivo is still unclear. Our results showed that systolic cardiac dysfunction in Tg MAOA mice was strongly correlated with oxidative stress induced premature senescence of cardiac stromal cells favoring the recruitment of CCR2+ monocytes and the installation of cardiac inflammation. Understanding the interplay between oxidative stress induced premature senescence and accelerated cardiac dysfunction will help to define new molecular pathways at the crossroad between cardiac dysfunction and accelerated aging, which could contribute to the increased susceptibility of the elderly to HF

Shear Stress-Induced Alteration of Epithelial Organization in Human Renal Tubular Cells.

Tubular epithelial cells in the kidney are continuously exposed to urinary fluid shear stress (FSS) generated by urine movement and recent in vitro studies suggest that changes of FSS could contribute to kidney injury. However it is unclear whether FSS alters the epithelial characteristics of the renal tubule. Here, we evaluated in vitro and in vivo the influence of FSS on epithelial characteristics of renal proximal tubular cells taking the organization of junctional complexes and the presence of the primary cilium as markers of epithelial phenotype. Human tubular cells (HK-2) were subjected to FSS (0.5 Pa) for 48 h. Control cells were maintained under static conditions. Markers of tight junctions (Claudin-2, ZO-1), Par polarity complex (Pard6), adherens junctions (E-Cadherin, β-Catenin) and the primary cilium (α-acetylated Tubulin) were analysed by quantitative PCR, Western blot or immunocytochemistry. In response to FSS, Claudin-2 disappeared and ZO-1 displayed punctuated and discontinuous staining in the plasma membrane. Expression of Pard6 was also decreased. Moreover, E-Cadherin abundance was decreased, while its major repressors Snail1 and Snail2 were overexpressed, and β-Catenin staining was disrupted along the cell periphery. Finally, FSS subjected-cells exhibited disappeared primary cilium. Results were confirmed in vivo in a uninephrectomy (8 months) mouse model where increased FSS induced by adaptive hyperfiltration in remnant kidney was accompanied by both decreased epithelial gene expression including ZO-1, E-cadherin and β-Catenin and disappearance of tubular cilia. In conclusion, these results show that proximal tubular cells lose an important number of their epithelial characteristics after long term exposure to FSS both in vitro and in vivo. Thus, the changes in urinary FSS associated with nephropathies should be considered as potential insults for tubular cells leading to disorganization of the tubular epithelium